I-.il.-.'  -.  ptotnber  7,  1912, 


U.  S.  DEPARTMJ  NT  OF  AGRICULTURE, 

\mm  \  I    INDUSTRY      R  LLI  rw  IH. 

A.  I>.  Ml  I  -*u. 


A  STUDY  OF  THE  GASES  OF 
EMMENTAL  CHEESE. 


BY 


WILLIAM   MANSFIELD    CLARK,   Ph.  D., 
Chemist,  Dairy  Division, 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 

1912. 


U.  S.  DEPARTMI  NT  OF  AGRICULTURE, 

WIMAl    INDI  I    I 


A  STUDY  OF  THE  GASES  OF 

EMMENTAL  CHEESE. 


BY 


WILLIAM   MANSFIELD    CLARK,   Ph.  D., 

Chemist,  Dairy  Division. 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 

1912. 


THE  BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  Melyin. 

Assistant  Chief:  A.  M.  Farrington. 

Chief  Cleric:  Charles  C.  Carroll. 

Animal  Husbandry  Division:  George  M.  Rommel,  chief. 

Biochemic  Division:  M.  Dorset,  chief. 

Dairy  Division:  B.  H.  Rawl,  chief. 

Field  Inspection  Division:  R.  A.  Ramsay,  chief. 

Meat  Inspection  Division:  Rice  P.  Steddom,  chief. 

Pathological  Division:  John  R.  Mohler,  chief. 

Quarantine  Division:  Richard  W.  Hickman,  chief. 

Zoological  Division:  B.  H.  Ransom,  chief. 

Experiment  Station:  E.  C.  Schroeder,  superintendent. 

Editor:  James  M.  Pickens. 

DAIRY  DIVISION. 

B.  H.  Rawl,  Chief 

Helmer  Rabild,  in  charge  of  Dairy  Farming  Investigations. 
S.  C.  Thompson,  in  charge  of  Dairy  Manufacturing  Investigations. 
L.  A.  Rogers,  in  charge  of  Research  Laboratories. 
Ernest  Kelly,  in  charge  of  Market  Milk  Investigations. 
Robert  McAdam,  in  charge  of  Renovated  Butter  Inspection. 
2 


ADDITIONAL  COPIES  of  this  publication 
-£±-  may  be  procured  from  the  Superintend- 
ent of  Documents,  Government  Printing 
Office,  Washington,  D.  C,  at  5  cents  per  copy 


LETTER  OF  TRANSMITTAL. 


U.  S.  Department  op  A.grioulti  be, 

Bureau  of  Animal  [ndustry, 

Washington,  D.  <\,  April 23,  191$, 
Sir:  I  have  the  honor  to  transmit,  and  to  recommend  for  publi- 
cation in  the  bulletin  series  of  the  bureau,  the  accompanying  manu- 
script entitled  "A  Study  of  the  Gases  of  EmmentaJ  Cheese/1  by  Dr. 

William  Mansfield  Clark,  chemist  in  the  Dairy  Division. 

The  so-called  "eyes"  in  Swiss  cheese  are,  as  is  well  known,  its  most 
prominent  characteristic,  and  its  commercial  value  is  largely  depend- 
ent upon  the  proper  size  and  spacing  of  these  eyes.  Furthermore, 
much  depreciation  in  the  value  of  this  popular  variety  of  cheese,  in 
both  the  domestic  and  foreign  kinds,  is  known  to  exist  because  of 
defects  in  eye  formation.  The  experimental  work  herein  described 
concerns  the  chemical  contents  of  these  eyes,  and  although  consider- 
able work  has  been  done  in  Europe  with  the  object  of  discovering  the 
cause  of  eye  formation,  there  has  lutherto  been  no  investigation  made 
of  the  gases  which  are  immediately  concerned  in  the  process.  Dr. 
(lark's  studies  are  therefore  calculated  to  be  of  value  to  the  scientific 
as  well  as  the  practical  side  of  the  industry. 

Respectfully, 

A.  D.  Melvix, 

Chief  of  Bureau. 

Hon.  James  TTilsox, 

Secretary  of  Agriculture. 

3 


CONTENTS. 


Page. 

Introduction 7 

Description  of  apparatus  and  methods  of  collecting  the  pases 9 

Method  1 9 

Method  II 10 

Method  of  analysis 11 

Discussion  of  the  analyses 12 

A 1 1»  »rpt  i(  »n  of  oxygen 18 

The  permeability  of  cheese  to  gases 20 

Nitrogen  dissolved  in  curd 

Does  nitrogen  originate  in  situ? 25 

Relation  between  carbon  dioxid  and  volatile  acids L'G 

Summary 31 

References  to  literature 32 

5 


ILLUSTRATIONS. 


Page. 

Fig.  1.  Apparatus  for  collecting  gas  from  the  eyes  of  Swiss  or  Emmental  cheese. .  8 

2.  Apparatus  for  pumping  gas  from  cheese 10 

3.  Apparatus  for  studying  the  absorption  of  oxygen  by  cheese 14 

4.  Device  for  ascertaining  permeability  of  cheese  to  gases 20 

5.  Apparatus  for  determining  amount  of  nitrogen  in  curd 24 

6 


A  STUDY  OF  THE  GASES  OF  EMMENTAL  CHEESE. 


INTRODUCTION. 

The  "eyes"  of  Swiss  or  Emmenta]  cheese  arc  its  most  striking 
characteristic.  Their  formation  is  a  fascinating  subject  to  the  bio- 
logical chemist,  because  of  a  supposed  localization  of  reactions  gen- 
erating considerable  quantities  of  gas,  and  because  of  the  produc- 
tion of  a  plasticity  among  the  colloids  of  the  cheese,  winch  makes 
possible  the  peculiar  mold  of  the  cavities. 

To  the  cheese  maker  the  formation  of  the  "eyes"  is  a  matter  of 
great  importance,  since  their  size  and  proper  spacing  determine  in 
large  measure  the  commercial  value  of  the  cheese.  In  certain  dis- 
tricts of  Wisconsin  visited  by  the  writer  the  dealers  rely  ah 
entirely  upon  these  features,  and,  shortly  after  the  eyes  have  reached 
their  proper  development,  relieve  the  maker  of  further  care.  The 
American  makers  of  Swiss  cheese  are,  therefore,  unable  to  attend 
to  their  cheeses  in  that  mellow  old  age  upon  which  so  much  of  the 
fine  flavor  of  a  true  Emmental  cheese  depends.  However  much  this 
quick  marketing  is  to  be  deprecated,  the  fact  remains  that  it  raises 
the  relative  importance  of  the  eye  formation  and  adds  significance 
to  whatever  knowledge  can  be  gained  concerning  the  process. 

Some  years  ago  Baehler,1  a  cited  by  Jensen,11  estimated  that  25 
per  cent  of  the  cheeses  made  in  Switzerland  were  considerably  reduced 
in  value  because  of  imperfect  eye  formation.  How  far  this  enormous 
loss  has  been  lessened  in  recent  years  as  a  result  of  scientific  ally  con- 
trolled manufacture  can  not  be  said,  but  in  this  country,  where  large 
numbers  of  Swiss  are  still  using  the  antiquated  methods  of  their  fore- 
fathers, Bachler's  estimate  is  probably  not  too  high.  The  wide  dif- 
ference in  market  price  between  domestic  and  imported  Swiss  cheese 
bears  out  this  statement. 

Considerable  work  has  been  done  in  Europe  in  the  effort  to  uncover 
the  cause  of  eye  formation,  and,  through  the  labors  particularly  of 

o  The  reference  figures  relate  to  the  list  of  references  to  literature  at  end  of  bulletin. 

7 


8 


STUDY  OF   GASES   OF  EMMENTAL   CHEESE. 


Von  Freudenreich  and  Jensen,  a  well-founded  theory  has  been  proposed 
which  will  be  discussed  later.  No  one,  however,  has  made  a  study  of 
the  gases  winch  are  themselves  the  immediate  cause  of  the  eye  forma- 
tion, and  it  was  with  the  hope  that  such  a  study  might  furnish  valu- 
able data  that  the  research  herein  described  was  undertaken.  If 
nothing  more  is  demonstrated  than  the  composition  of  the  gas  in 


mF 


Fig.  1.— Apparatus  for  collecting  gas  from  the  eyes  of  Swiss  or  Emmental  cheese. 

the  eyes,  this  alone  justifies  the  work,  for  the  extensive  researches  on 
the  eye  formation  in  Emmental  cheese  have  led  to  but  one  conclu- 
sion that  can  be  called  positive,  and  that  is  that  a  final  explanation 
will  be  reached  only  when  every  phase  of  the  subject  has  been  sub- 
mitted to  exact  quantitative  study. 


APPARATUS  AND  METH0D6.  9 

DESCRIPTION    OF   APPARATUS    AND    METHODS    OF    COLLECTING    THE 

GAS 

Tin*  collection  of  the  gas  in  the  eyes  by  cutting  the  cheese  undi 
bell  jar  filled  with  water,  as  waa  done  with  Edam  cheese  1  boul 

and  Ott  de  Vries,1  is  a  simple  and  valuable  method,  but  one  which 
is  hardly  to  be  called  ace u rate,  owing  to  the  high  solubility  of  certain 

e  in  water.     In  pi  act*  of  such  a  method  an  apparal  us  was  dei 
for  collecting  the  gas  over  mercury.    This  is  Bhown  in  figure  l,  the 
procedure  being  as  follows: 

METHOD    I. 

The  glass  cylinder  A   is  forced  a  short  distance  into    the   body  of 

the  cheese  until  it  is  firmly  held.     It  is  then  damped  in  position. 

Around  the  outside  the  cheese  is  cut  away  sufficiently  to  leave  ;i 
channel  into  wlrich  mercury  moistened  with  mercuric  chloiid  soli  it  inn 
is  poured.  This  forms  a  seal  preventing  entrance  of  air.  The  head 
of  the  shaft  B  is  now  resting  on  the  surface  of  the  cheese.  Through 
its  capillary  mercury  is  run  into  the  cylinder,  displacing  the  air  until 
it  finally  runs  out  of  the  side  arm  D  and  up  through  the  annular 
space  between  the  shaft  and  the  shoulder  of  the  cylinder.  The  short 
length  of  thick  rubber  tubing  at  E  is  then  very  tightly  bound  with  a 
rubber  band,  leaving  mercury  in  the  small  cup  above,  and  thus 
effectually  closing  tins  opening  against  the  entrance  of  air.  When 
the  cylinder  and  side  arm  are  thus  completely  filled  with  mercury,  a 
receptacle  filled  with  mercury  is  brought  over  the  end  of  the  side 
arm  (in  a  mercury  trough,  of  course)  and  serves  to  retain  the  col- 
lected gas  until  the  time  of  the  analysis.  After  these  preparations 
the  shaft  is  pushed  down  into  the  cheese.  When  it  punctures  an 
eye  this  can  readily  be  felt.  Since  the  head  of  the  shaft  is  larger 
than  the  shank,  there  is  left  an  annular  space  for  the  escape  of  the 
gas.  This  gas  is  displaced  from  the  eye  partially  by  the  mercury  of 
the  cylinder,  winch  finds  its  way  to  the  lower  level,  but  more  largely 
by  the  mercury  which  runs  in  through  the  capillary  in  the  shaft.  The 
exit  of  this  is  prevented  from  becoming  clogged  with  cheese  by  care- 
fully blowing  it  out  just  behind  the  head,  as  shown  in  the  diagram. 
When  the  gas  is  displaced  from  the  eye  it  is  displaced  from  the 
cylinder  into  the  receiver  by  continuing  to  run  in  mercury  through 
the  shaft  from  the  reservoir  C.  Between  this  reservoir  and  the  shaft 
is  placed  a  bulb  which  prevents  the  mercury  from  sweeping  in  bub- 
bles of  air. 

In  the  samples  of  gas  collected  with  this  apparatus  seldom  was  more 
than  a  trace  of  oxygen  found.     This  in  itself  shows  that  the  gas  was 
obtained  without  contamination  by  air. 
42208°— Bull.  151—12 2 


10 


STUDY   OF    GASES   OF   EMMENTAL   CHEESE. 


METHOD   II. 

For  the  collection  of  gas  from  "pinholes"  the  foregoing  apparatus 

was  of  little  use  except  in  one  instance  to  be  mentioned  later.     To 

collect  the  gas  from  this  form  of  hole,  as  well  as  the  gas  in  the  body 

of  the  cheese,  the  apparatus  shown  in  figure  2  was  used,  as  follows: 

Samples  of  cheese  taken  with  a  trier  were  introduced  into  the 

glass  cylinder  A.  The  rubber 
stopper  at  B,  attached  to  the 
mercury  vacuum  pump  with  or 
without  the  intermediate  connec- 
tion C,  was  forced  in  securely  and 
protected  from  leakage  by  the 
mercury  seal.  Upon  raising  the 
leveling  bulb  D  the  cheese  was 
flooded  with  mercury  and  the  sur- 
rounding air  was  forced  over  into 
the  pump  until  the  mercury  stood 
at  the  stopcock  E.  To  prevent 
bubbles  of  air  being  trapped  under 
the  cheese  the  lower  ends  of  the 
plugs  were  sharply  beveled.  Bub- 
bles of  air  of  course  adhered  to  the 
rough  surface  of  the  cheese  and  its 
smaller  exposed  cavities.  This 
error  is  inherent  in  the  method, 
but  was  reduced  by  suddenly  drop- 
ping the  leveling  bulb  with  the  stop- 
cock E  closed,  and  then  driving  the 
air,  which  had  expanded  into  the 
vacuum,  past  the  open  stopcock. 
The  glass  tube  with  its  trap 
which  connects  A  with  the  level- 
ing bulb  was  made  sufficiently 
long  so  that  D  might  be  lowered 
the  barometric  distance  below  A, 
and  thus  leave  the  cheese  exposed 
to  a  fairly  high  vacuum  even  be- 
fore the  pumping  commenced.  After  exhausting  the  pump  up  to 
E  this  cock  was  opened,  and  the  gas  pumped  from  the  cheese  and 
delivered  into  a  receiver. 

The  mercury  pump  used  in  this  as  in  other  operations  to  be  de- 
scribed later  was  AntropofFs  modification  of  the  Topler.  A  full 
description  of  the  pump  and  its  appurtenances  will  appear  in  the 
account  of  another  investigation. 


Fig.  2.— Apparatus  for  pumping  gas  from  cheese. 


Ml   I  HOD    01      \N  W  \  SIS. 


11 


METHOD  OF  ANALYSIS. 

The  gas  was  analyzed  with  a  special  Bet  of  burettea  and  pipe! 
designed  for  the  analysis  of  small  quantities  of  gas  produced   by 
bacteria.     A  few  of  the  Bret  analyses  urn-  made  with  a  burette 
dally  designed  for  volumes  as  low  as  0.5  c.  <-.     In  all  the  ana] 
the  confining  liquid  was  mercury,  and  use  wna  made  of  a  device  for 
extremely  accurate  sepi  ration  of  gas  from  absorbent. 

Thirty-three  per  cent  potassium  hydrozid  solution,  in  quantities 
appropriate  for  the  volume  of  gas  analyzed,  served  as  absorbent  for 
carbon  dioxid.  Hydrogen  Bulphid,  after  preliminary  qualitative 
tests,  was  assumed  to  be  absent,  although  it  is  of  course  possible 
that,  if  present  originally  in  the  gas,  it.  may  have  been  taken  up  by 
the  mercury.  That  any  of  this  gas  occurs  in  the  eyes  is,  however, 
very  improbable,  for  its  odor  was  never  detected.  For  hydn 
sulphid  and  mercaptans  the  nose  is  many  times  more  sensitive  than 
e  spectroscope  for  sodium,1  and  unless  the  other  and  milder 
odors  of  Swiss  cheese  exercise  a  Mirprisingly  intense  hindrance  to  the 
detection  of  hydrogen  sulphid  and  mercaptans  we  may  justly  say 
that  these  vapors  were  absent.  With  Nessler's  reagent  very  slight 
traces  of  ammonia  were  detected.  For  oxygen  alkaline  pyrogallol 
or  long-continued  contact  with  phosphorus  was  used.  Combustible 
gases  were  estimated  in  several  ways.  Explosion  with  oxygen,  in 
the  presence  of  electrolytic  gas  when  necessary,  was  used  in  several 
instances.  For  one  case  combustion  with  a  platinum  sponge  was 
tried.  For  the  small  percentages  of  combustible  gases  found  the 
method  of  Dennis  and  Hopkins5  was  found  to  be  the  most  satis- 
factory. This  consists,  essentially,  in  leading  the  gas  slowly  into  a 
measured  volume  of  oxygen  and  there  burning  it  slowly  and  quietly 
with  a  platinum  wire  heated  by  an  electric  current. 

Table  1. — Analyses  of  gas  collected  by  puncturing  apparatus  from  eyes  of  Swiss  (Em- 
mental)  cheese — Method  I. 


Desig- 
nation 

of 
cheese. 

Total 
vol- 
ume of 
gas  col- 
lected. 

Contraction- 

Composition. 

Due  to 
absorp- 
tion 
with 
KOH. 

Due  to 
absorp- 
tion 
for  02. 

Due 
to 
com- 
bus- 
tion. 

Hydro- 
COj.       Oj.         car- 
bons. 

Bb. 

x2. 

Description  of  cheese. 

a 

C.c. 

0.96 

2.73 

1.66 

4.77 

1.25 

/    3.44 

\    4.02 

15.24 

/    7.56 

1     4.99 

/  14.47 

\    9.99 

5.42 

4.96 

C.c. 
0.55 
2.29 
1.11 
2.44 
1.00 
2.23 
2.52 

13.77 
6.14 
4.04 

12. 91 
8.95 
3.04 
2.35 

C.c. 

C.c. 

Per 

cent. 
57.3 
83.9 

Per 

cent. 

Per 

cent. 

Per 

cent. 

Per 

cent. 

Imported,  eyes  normal. 
Do. 

b 

0.00 
.01 
.02 
.00 
.00 

"".'66 

.02 
.03 
.03 
.01 
.02 
.01 

o.'io' 

.02 
.00 
.60 
.45 
.76 
.43 
.30 
.00 
.09 
.06 
3.63 

0.0 

c 
d 
e 

f 

g 
h 

i 
39.61 

66.9 
51.2 
80.0 
64.8 
62.7 
90.4 
81.2 
80.9 
89.2 
89.5 
56.0 
47.4 

Trace. 

Trace. 
0.0 
0.0 

""""6."  6" 

Trace. 
0.6 
0.2 

Trace. 

Trace. 

Trace. 

(?W*n 

0.0 
0.0 
0.0 
0.0 
Trace? 
0.0 
0.0 
0.0 
0.0 
0.0? 
0.0 

4.00 

Trace? 

0.0 

11.6 
7.5 
3.3 
3.7 
4.0 
0.0 
0.6 

Tne  ■? 

29.1 
18.8 

20.0 
23.6 
29.8 

6.3 
1.5.1 
14.5 
10.6 

9.8 
44.0 

3.8 

Imported,  eyes  (?). 

Imported,  eyes  normal. 
Do. 
\  Domestic,    eyes     thickly 
/    crowded. 

Do. 
\ Imported,    eyes    thickly 
/     crowded. 

^Excellent  Imported,  eyes 
J     very  regular. 

Imported,  large  hole. 

Very  gassy  in  press. 

12 


STUDY   OF    GASES   OF   EMMENTAL   CHEESE. 


The  analyses  of  the  gas  collected  Jby  Method  I  are  given  in  Table  1, 
and  of  that  collected  by  Method  II  in  Table  2.  All  volumes  are  for 
0°  C.  and  760  mm.  When  the  gases  were  collected  from  a  cheese  pro- 
cured at  the  market,  a  sufficiently  large  slice  was  purchased  to  pre- 
vent undue  exposure  of  the  eyes,  and  this  was  carried  immediately 
the  short  distance  to  the  laboratory,  and  the  gas  at  once  collected. 
In  most  cases  the  shaft  punctured  or  grazed  more  than  one  eye,  so 
that  the  analysis  gives  the  true  average  for  several  eyes. 

Table  2. — Analyses   of  gas  collected  by  pumping  from  Swiss   (Emmental)   cheese — 

Method  II 


No  of 

Time 
pump- 
ing. 

Total 
gas  col- 
lected. 

Weight 

of  cheese 

evacuar 

ted. 

Amount 
of  gas 

per  100 
grams  of 

cheese. 

Composition. 

cheese. 

COj. 

o2. 

Ha. 

Nt. 

Description  of  cheese. 

3 

Hours. 
20 

20 " 

20 " 

C.c. 
2.36 

2.31 
6.41 
3.20 
13.60 

Grams. 

C.c. 

Per 
cent. 
76.3 

77.5 
80.8 
50.6 
84.5 

Per 

cent. 

1.7 

2.6 
2.0 
1.0 
2.2 

Per 

cent. 

0.0 

0.0 
0.0 
0.0 
0.0 

Per 
cent. 
22.0 

19.9 
17.2 
48.4 
13.3 

Almost  blind.    Several  small 

39-45 

holes,  either  pinholes  or  in- 
hibited eyes. 
Do. 

39-11-2 

Do. 

46-4-1 
W2 

50 
53 

6.40 
25.7 

Do. 

Fine  domestic  cheese  just  be- 
ginning eye  development. 

DISCUSSION  OF  THE  ANALYSES. 

If  the  values  obtained  in  this  study  of  the  gases  found  in  the  eyes 
of  Swiss  cheese  are  compared  with  the  values  obtained  by  Boekhout 
and  Ott  de  Yries  2  for  the  gases  in  Edam  cheese,  it  is  seen  that  the 
latter  obtained  much  lower  percentages  of  carbon  dioxid  and  corre- 
spondingly higher  percentages  of  nitrogen.  The  explanation  becomes 
apparent  when  it  is  remembered  that  Boekhout  and  Ott  de  Vries 
collected  the  gas  over  water,  while  in  this  investigation  it  was  collected 
over  mercury.  The  two  methods  were  compared  in  the  case  of  cheese 
h,  as  follows: 


Method. 


Collection  over  mercury 
Collection  over  mercury 
Collection  over  water. . . 


C02. 


Per  cent. 
81.2 
80.9 
34.8 


02. 


Per  cent. 

Trace. 

0.6 

1.9 


H2. 


Per  cent. 
3.7 
4.0 
1.9 


N2. 


Per  cent. 
15.1 
14.5 
61.4 


This  result  is  what  might  have  been  expected,  namely,  an  absorp- 
tion of  much  carbon  dioxid  and  a  little  hydrogen  by  the  water,  and, 
in  return,  an  increase  in  the  amount  of  oxygen  as  well  as  an  increase 
in  percentage  of  nitrogen.  Boekhout  and  Ott  de  Vries  have  them- 
selves called  attention  to  this,  and  claim  only  qualitative  value  for 
their  results.     The  types  of  holes  from  which  they  isolated  gas  were 


small  cracks  corresponding  to  the  Emmental 
holes,  and  large  cracks  termed  "knijpers." 


riszler,"  small  round 


UB01  BfUOS    01    \  \  \i  \  1  8 

Qualitatively  the  composition  of  the  gases  was  the  isme,  namely, 
carbon  dioxid,  hydrogen,  uitrogenfand  oxygen.  (  m'  these  thej  elimi- 
nated oxygen  as  due  to  contamination.  In  bh<  of  the  "knij- 
pers,n  or  large  cracks,  52  to  249  c.  <•.  of  gas  were  collected  instead  of 
,")  to  22  c.  c  as  in  the  case  of  the  smaller  holes.  Assuming  thai  the 
same  volume  of  water  was  used,  we  would  expect  a  truer  value  to  be 
obtained  for  the  analysis  of  the  larger  volumes,  in  which  case  the 
attention  is  Btruck  by  the  large  percentage  of  hydrogen.  The  signifi- 
cance of  this  will  become  apparent  when  the  results  on  Brnmental 
cheese  have  been  assembled. 

li  is  clear  from  the  analyses  of  gas  found  in  Emmenta]  cheese 
thai  carbon  dioxid  and  nitrogen  are  the  chief  constituents  of  tin 
found  in  normal  eyes.  The  oxygen  in  most  cases  is  hardly  more  than 
would  be  expected  to  come  from  the  minute  bubbles  or  Burface 
layers  which  adhere  to  the  glass  walls  of  the  apparatus.  To  what 
gas  the  contraction  after  explosion  with  oxygen  is  to  be  ascribed  is  a 
difficult  question  to  settle.  In  some  cases,  where  the  contraction 
was  sufficiently  Large  to  justify  further  absorption  with  potassium 
hydroxid,  the  absence  of  any  further  contraction  in  volume  justifies 
the  conclusion  that  the  combustible  gas  was  cluefly  hydrogen.  In 
other  cases  the  small  contraction  might  have  been  clue  to  any  one  of 
a  number  of  gaseous  combustions. 

For  further  information  it  was  decided  to  examine  specimens  of 
spectroscopically.  The  gas  freed  from  carbon  dioxid  end  possible 
oxygen  was  passed  over  phosphorus  pentoxid  into  a  dry,  exhausted 
Pliickcr  tube.  The  discharge  of  an  induction  coil  was  then  pas 
between  aluminum  terminals,  and  the  spectrum  observed  with  a 
prism  spectroscope.  At  the  same  time  comparison  was  made  with  the 
spectrum  of  a  similar  tube  containing  pure  hydrogen.  Minute  traces 
of  hydrogen  are  to  be  expected  when  metal  terminals  are  used,  but, 
with  the  low  resolving  power  of  the  spectroscope  employed,  the  nitro- 
gen spectrum  so  obscured  the  possibly  present  red  line  of  hydrogen 
that  it  was  not  observed  with  specimens  of  pure  nitrogen.  A  known 
sample  of  nitrogen  containing  about  0.05  per  cent  of  hydrogen  gave 
a  brilliant  hydrogen  spectrum,  whose  intensity  could  be  made  more 
sharp  at  the  expense  of  the  nitrogen  spectrum  by  suitable  varying  of 
the  pressure.15  The  recognition  of  0.05  per  cent  of  hydrogen  was 
therefore  assured. 

A  small  experimental  cheese,  which  had  begun  an  apparently  nor- 
mal eye  formation  and  then  ceased  entirely,  was  pumped  out  by 
Method  II  and  its  gas  submitted  to  spectroscopic  examination. 
Slight  evidences  of  hydrogen  were  observed.  Samples  of  gas  taken 
from  cheeses  which  yielded  3  per  cent  of  combustible  gas  gave  very 
brilliant  evidences  of  hydrogen. 


14 


STUDY  OF   GASES   OF  EMMENTAL   CHEESE. 


In  samples  of  gas  taken  from  the  normal  eyes  of  two  cheeses  pur- 
chased on  the  market  no  hydrogen  line  was  observed,  nor  was  the 
hydrogen  spectrum  observed  in  the  gases  of  a  normal  cheese  evolved 
during  the  period  of  its  maximum  eye  formation. 

These  results,  though  not  extensive, 
are  sufficient  to  show  that  hydrogen 
plays  no  role  in  the  formation  of  normal 
eyes,  provided  we  assume  that  any  hy- 
drogen formed  has  not  escaped  collection 
by  rapidly  diffusing  through  the  cheese. 
To  make  sure  of  this  point  the  following 
experiments  were  conducted: 

Two  cheeses  purchased  in  Wisconsin 
were  found  to  be  developing  normal  eyes. 
These  eyes,  though  too  thickly  scattered 
for  the  modern  market  standard,  would 
have  been  declared  typical  some  years 
ago.  "When  each  cheese  was  apparently 
at  the  height  of  its  eye  formation,  plugs 
were  taken,  and  introduced  into  the  tube 
A,  figure  3,  without  that  part  illustrated 
at  the  side  and  lettered  G,  F,  and  E.  To 
guard  as  far  as  possible  against  infection 
in  transference  the  trier  was  flamed,  and 
the  tube  was  sterilized  at  170°  C,  with 
cotton  plugs  at  B  and  C.  After  intro- 
ducing the  plugs  of  cheese  they  were  fol- 
lowed by  the  flamed  cotton  plug  and  then 
a  rubber  stopper  dipped  in  hot  rubber 
cement.  The  stopper  was  forced  in  and 
held  in  place  till  the  cement a  had  cooled, 
when  several  layers  of  the  same  cement 
were  added  to  the  exterior.  This  made 
a  thoroughly  gas-tight  seal.  The  capil- 
lary end  was  now  attached  to  the  mercury 
pump  by  means  of  securely  tied  rubber 
tubing  completely  covered  with  a  mercury  seal.  Then  the  tube  was 
exhausted. 

Forty-six  grams  from  one  of  the  Wisconsin  cheeses  were  exhausted 
for  two  hours,  during  which  time  it  continued  to  give  off  small 
quantities  of  gas.  The  pressure  was  finally  reduced  to  2  mm.  (meas- 
ured on  a  McLeod  gauge).     The  stopcock  D  was  then  closed,  and  the 

a  The  cement  was  made  by  heating  rosin  several  days  with  as  much  fine-grade  rubber  as  it  would  dissolve. 
Dr.  Nutting,  of  the  Bureau  of  Standards,  who  kindly  furnished  the  receipt,  stated  that  he  had  used  this 
cement  in  refined  vacuum  work  with  entire  satisfaction. 


£ 


Fig.  3.— Apparatus  for  studying  the  ab- 
sorption of  oxygen  by  cheese. 


DSEM  OT98IOB   01   UTALTBMl  1  .r> 

tube  allowed  to  remain  in  oonnection  with  the  |  >n m | >  overnight. 
The  next  morning  the  pumping  was  resumed)  and  b  pressure  <>f  2  mm. 
sgain  obtained.  The  gaa  which  had  collected  overnight  amounted 
to  7.23  c.  c.j  -V  T.  I*.     [ta  analysis  follows: 

inal  volume 

Eb   idue  after  absorption  with  KOH. .  



( »w  -n  added  up  to »2 

Volume  after  oombustioD  with  heated  platinum  spiral. . .  2.  2fl 

Contraction 

The  tulx1  was  then  Bealed  off  in  a  blowpipe  at  the  constriction  II 
and  kept  for  si\:  days  at  25  '  C.  To  collect  the  gas  from  this  Bealed 
tube  the  following  method  was  used.  The  capillary  tip  of  the 
was  scratched  with  a  diamond}  and  then  pushed  up  into  the  tube 
Leading  from  the  pump  as  at  C,  figure  3.  Connection  was  made  with 
a  rubber  tube  securely  tied  and  covered  with  a  mercury  seal.  Having 
exhausted  the  pump  up  to  the  tip  of  the  seal,  the  tube  was  turned 
slightly  and  sharply.  The  tip  was  broken  at  the  scratch,  and  com- 
munication established  between  A  and  the  pump. 

The  gas  thus  collected  at  the  end  of  six  days  amounted  to  10.12 
c.  c.  The  tube  was  allowed  to  stand  connected  with  the  pump  over- 
night, after  which  an  additional  2.75  c.  c.  of  gas  were  collected. 

These  two  volumes  were  united  and  analyzed  99.3  per  cent  carbon 
dioxid.  The  residue  was  hardly  sufficient  to  justify  further  analysis. 
It  was  made,  however,  and  a  minute  contraction  observed,  which  was 
hardly  more  than  the  experimental  errors  of  transference. 

Forty-five  grams  of  "the  second  Wisconsin  cheese  submitted  to  the 
same  procedure  as  described  above  gave  the  following  data: 

c.c. 

Gas  collected  on  first  standing  overnight : 11. 48 

Residue  alter  absorption  with  KOII 22 

C02 11.26 

Residue  after  absorption  with  phosphorus .22 

Oxygen  added  up  to a  2. 41 

Volume  after  combustion  with  heated  platinum  spiral 2.  39 

Tube  sealed  off  and  incubated  six  days  at  25°  C. 

J  C.c. 

Gas  collected  after  G  days 9.  29 

Residue  after  absorption  with  KOH 16 

C02 9. 13 

Oxygen  added  up  to °1.  91 

Volume  alter  combustion  with  hot  platinum  spiral 4.  85 

Gas  collected  after  again  standing  overnight 5.  33 

Residue  after  absorption  with  KOH Trace. 

a  This  comparatively  large  volume  was  made  necessary  because  of  the  disadvantageous  form  of  the 
Dennis-Hopkins  pipette  used. 


16  STUDY   OF    GASES   OF   EM  MENTAL   CHEESE. 

In  the  above  analyses  the  contraction  due  to  combustion  was  s6 
small  that  further  analyses  to  determine  the  products  of  combustion 
were  impracticable.  Nor  was  it  necessary,  for,  even  if  the  contraction 
Were  due  to  but  one  gas,  for  example  hydrogen,  the  amount  was  such 
that  this  gas  may  be  said  to  be  without  significance  in  the  formation 
of  eyes.  Doubtless  the  contraction  was  in  reality  due  to  volatile 
organic  bodies.  The  above  experiments  show  that  when  all  the  gas 
from  an  actively  gas-producing  region  is  collected  no  significant 
amount  of  hydrogen  is  found,  and  thereby  the  contention  is  refuted 
that,  in  the  analysis  of  gas  in  the  eyes,  hydrogen  escaped  detection 
because  of  its  rapid  diffusion  out  through  the  cheese. 

Pains  were  taken  in  these  studies  to  make  a  strenuous  hunt  for 
hydrogen  for  the  following  reason:  In  Emmental  cheese  there  is 
what  Duclaux  has  termed  the  "initial  fermentation"  during  which 
the  sugar  inclosed  in  the  curd  undergoes  bacterial  decomposition. 
Several  of  the  earlier  workers  on  this  cheese  thought  it  was  the  gaseous 
fermentation  of  tins  sugar  which  caused  the  development  of  eyes.  If 
so,  one  would  expect  to  find  the  gas  composed  of  a  large  percentage  of 
hydrogen,  since  hydrogen  is  a  characteristic  product  in  the  fermenta- 
tion of  sugars  by  bacteria.  This  deduction  is  of  course  not  rigid,  but, 
from  our  present  knowledge  of  the  gaseous  fermentation  of  sugars  by 
bacteria,  it  is  highly  probable. 

Jensen  "  in  1898  pointed  out  clearly  that  the  gaseous  fermenta- 
tion of  sugar  must  not  be  looked  upon  as  in  any  way  directly  connected 
with  the  production  of  normal  eyes  in  Emmental  cheese.  He 
found  no  trace  of  sugar  in  a  cheese  five  days  old,  although  the  normal 
eye  formation  had  not  yet  begun.  This  confirms  the  analyses  made 
by  various  authors.  Jensen  cited  Klenze  13  as  stating  that  the  sugar 
disappears  in  48  hours.  But,  while  the  sugar  disappears  rapidly, 
normal  eyes  seldom  begin  to  develop  before  the  eighth  day,  and  reach 
the  height  of  their  development  long  after  every  trace  of  sugar  has 
disappeared. 

These  facts  alone  demonstrate  that  the  eye  formation  does  not 
depend  upon  the  presence  of  sugar.  Additional  reason  for  so  believ- 
ing is  found  in  the  results  herein,  in  so  far  as  the  absence  of  hydrogen 
in  the  gas  indicates  an  absence  of  gaseous  sugar  fermentation. 

But  it  also  follows  from  this  reasoning  that  when  a  gaseous  fermen- 
tation occurs  while  sugar  is  still  present  in  the  cheese,  hydrogen  is  to 
be  expected.  Such  a  fermentation  frequently  occurs  while  the  cheese 
is  in  press.  Fortunately  a  cheese  was  obtained  (No.  39-61)  which  was 
known  to  have  given  marked  signs  of  gas  while  under  press.  From 
this  cheese  gas  was  collected  by  the  previously  described  Method  I, 
with  the  following  analysis : 

Total  volume  of  gas  collected Cubic  centimeters. .  4.  96 

Residue  after  absorption  with  KOH do 2.  61 

C02 do....  2.35 

Residue  after  absorption  with  phosphorus do 2.  60 


t)lSCt786lON   09    \  n  u  \  17 

Oxygen  added  tip  to.  Oubic  centimeten       I  1 1 

Volume  liter  combustion  frith  platinum  spiral 

Contraction do 

[due  after  absorption  with  K<  >i I 

II >  drogen. .....  percent 

[JpoD  attempting  to  make  a  second  puncture  the  mercury  broke 
through  into  the  hole  previously  made    The  cheese  was  then  opened, 

and  found  to  l>e  bo  Bpongy  that  the  walls  Beparating  the  individual 
cells  were  very  thin— too  thin  to  withstand  the  weight  <>f  mercury. 

To  obtain  a  second  sample  of  gas  f<>r  confirmatory  analysis  recourse 
was  had  to  Method  IT  of  collecting  gas,  previously  described.  A  high 
percentage  of  hydrogen  was  again  found. 

In  the  further  study  of  this  ca -<i  52  grams  of  the  cheese  were  intro- 
duced into  the  vacuum  tul>e  described  on  page  l  l  and  evacuated  to 
1  mm.  pressure.     There  collected  overnight  7. XI  <•.  c.  of  gas. 

Analysis: 

Total  volume 7.84 

Residue  after  absorption  with  COH 28 

Residue  after  absorption  with  phosphorus 27 

Oxygen  added  up  to ].0S 

Volume  after  combustion  with  platinum  spiral 99 

Contraction 09 

The  tube  was  then  sealed  off  and  kept  nine  days  at  25°  C.  Upon 
opening  it  and  pumping  out  the  gas  by  the  mot  hod  previously 
described  7.49  c.  c.  of  gas  were  collected.  The  residue  after  absorp- 
tion with  potassium  hydroxid  was  only  0.07  c.  c. 

It  is  therefore  apparent  that  the  production  of  hydrogen,  which 
was  very  active  while  the  cheese  was  in  press,  had  soon  ceased,  pre- 
sumably with  the  disappearance  of  the  sugar. 

The  occasional  occurrence  of  hydrogen  in  small  percentages,  as 
shown  in  the  table,  generally  accompanied  eyes  which  in  the  writer's 
judgment  were  not  typically  normal.  They  were  either  crowded  and 
distorted  or  associated  with  numerous  pinholes.  It  is  not,  perhaps 
incorrect  to  say  that  in  all  probability  there  had  occurred  in  these 
cases  a  slight  initial  gaseous  fermentation  of  the  sugar,  with  the  pro- 
duction of  hydrogen  which  lingered  to  contaminate  the  gas  of  the 
normal  fermentation. 

An  extremely  interesting  observation  was  made  in  the  case  of  cheese 
i.  (See  Table  1,  p.  11.)  This  was  an  excellent  imported  cheese  with 
large  and  perfectly  rounded  eyes,  well  spaced  in  a  body  of  fine  texture 
and  flavor.  In  the  first  analysis  of  the  gas  from  those  eyes  no  trace 
of  a  combustible  gas  was  found.  The  second  analysis  gave  0.0  per 
cent  of  hydrogen.  Upon  exposing  the  eyes  punctured  it  was 
observed  that  a  slight  crack  extended  to  within  a  centimeter  of  one  of 
the  eyes  punctured  on  the  second  collection.  This  crack  was  found 
to  lead  directly  to  a  hole  some  2  cm.  in  diameter,  the  irregular  and 
apparently  corroded  walls  of  which  proclaimed  it  distinctly  abnormal. 


18  STUDY  OF   GASES   OF  EMMENTAL   CHEESE. 

It  is  of  interest  to  note  that  in  the  case  of  cheese  j,  gas  was  obtained 
from  a  hole  the  size  of  one's  fist,  and  that  this  contained  practically 
no  hydrogen.  The  appearance  of  this  hole  was  that  of  a  strictly 
normal  eye  except  in  size. 

It  was  hoped  that  the  gas  of  a  typical  "blow  hole"  could  be  ob- 
tained. For  this  purpose  a  cheese  containing  such  a  hole  was  pur- 
chased in  Wisconsin.  When  it  arrived  at  the  laboratory  it  was  found 
that  the  cheesemaker  had  punctured  it. 

From  the  results  obtained  it  is  clear  that  there  are  at  least  two 
distinct  t}^pes  of  gas  formation.**  The  one  is  highly  detrimental,  and 
is  accompanied  with  hydrogen;  the  other  is  that  demanded  in  a  good 
Emmental  cheese.  One  is  dependent  upon  the  presence  of  sugar; 
the  other  occurs  in  the  absence  of  sugar. 

The  presence  of  hydrogen  in  considerable  quantities  in  the  gas  iso- 
lated from  Edam  cheese  by  Boekhout  and  Ott  de  Vries  is  very  sug- 
gestive of  a  gaseous  fermentation  of  sugar,  and  to  this  Jensen  "  has 
ascribed  the  formation  of  gas  holes  in  Edam  cheese. 

At  this  point  it  may  be  well  to  call  attention  to  a  source  of  error 
overlooked  by  various  investigators  in  their  attempts  to  establish 
the  cause  of  any  particular  gas  formation  in  cheese.  Frequent  exam- 
ples are  to  be  found  in  which  gas  production  by  bacteria  in  milk  is 
interpreted  to  mean  that  these  bacteria  can  produce  gas  in  cheese. 
Although  this  may  frequently  be  true,  it  must  nevertheless  be  remem- 
bered that  the  two  media  differ  not  only  in  chemical  constitution  but 
also  vary  greatly  in  physical  chemical  condition. 

Baumann,3  for  instance,  attributed  the  formation  of  eyes  in  hard 
cheeses  to  Bacillus  diatrypeticus  casei.  From  an  experiment  in 
which  this  bacillus  produced  in  milk  gas  containing  63  per  cent  of 
carbon  dioxid  and  the  remainder  almost  entirely  hydrogen,  Baumann 
concluded  that  the  gas  of  normal  as  well  as  faulty  eyes  is  carbon 
dioxid  and  hydrogen.  The  error  of  attributing  the  reactions  of  a 
bacillus  when  cultivated  in  milk,  which  contains  sugar,  to  cheese, 
which  after  the  initial  fermentation  contains  no  sugar,  is  so  evident, 
and  the  error  in  stating  that  the  gas  of  normal  eyes  contains  hydrogen, 
without  having  first  analyzed  this  gas,  is  so  evident,  that  Baumann's 
conclusions  might  be  left  unnoticed  at  this  late  date  were  they  not 
typical  of  several  found  in  the  more  recent  literature. 

ABSORPTION  OF  OXYGEN. 

In  all  the  analyses  no  appreciable  amount  of  oxygen  was  found. 
The  presence  of  large  percentages  of  nitrogen  with  this  absence  of 
oxygen  raises  the  question,  Does  air  diffuse  into  the  cheese  with  ab- 
sorption of  oxygen  ?     Evidence  of  an  active  absorption  of  oxygen  was 

a  This  does  not  preclude  there  being  a  number  of  distinct  fermentations  or  reactions  of  either  type. 


IB80BPTI0B    01     OXYGEN,  19 

accidentally  obtained.     In  attempts  tudy  the  produced 

in  sealed  tubes  a  faulty  Conn  of  tube  wraa  first  used,  \n.  1  i i « •  1  i  evidently 
leaked.  On  attempting  to  exhaust,  the  lowest  pressure  s/hicfa  could 
be  obtained  was  3.6  mm.  h  was  oon  a  certained  that  there  was  ho 
leak  in  the  pump,  but  a  leak  in  the  tube  was  Buspected.  Tin-  tube 
was  left  connected  with  the  pump  (connecting  stopcock  closed)  over 
night.  The  next  morning  37.20  c.o.  of  gas  whs  pumped  <mi .  The  first 
portion  of  19.15  c.  c.  gave  1.57  c.  c.  of  carbon  dioxid  and  2.21  c.  c.  of 
VII.    The  residue  was  lost  but  was  considered  to  be  nit  rogen.    The 

second  portion  was  then  pumped  out,  and  of  the  18.05  <•.  <•.  linn  col- 
lected there  were  4.85  c.  c.  of  carbon  dioxid,  1.45  c.  c.  of  oxygen,  and 
the  residue  entirely  nitrogen.  '1  lie  total  oxygen  amounted  to 3.66 o.c, 
which,  had  it  come  by  leakage,  would  have  indicated  an  entrance  of 
13.7  e.  e.  of  nitrogen.  There  \\  as  actually  found  24.12  c.  <•.  of  nitrogen. 
This  leaver  10.42  c.  c.  of  nitrogen  to  be  accounted  for.     The  carbon 

10  42 
dioxid  amounted  to  only  9. 1-  c.  c.  and,  since  the  ratio      '   v  is  much 
J  9.42 

larger  than  that  obtained  in  other  similar  pumpings  where  no  leak 

occurred,  it  was  suspected  that  oxygen  had  been  absorbed. 

To  definitely  determine  this  the  apparatus  shown  in  figure  3  was 

used.     With  plugs  of  cotton  at  B,  C,  and  in  the  bend  above  (1.  the 

tube  was  sterilized  at  170°  C.  Then  28.5  grams  from  one  of  the  Wis- 
consin cheeses  were  carefully  taken  with  trier  and  spatula  flamed  to 
prevent  contamination  as  far  as  possible,  and  the  plugs  introduced 
into  A  and  sealed  in  as  previously  described.  Mercury  was  drawn  up 
into  the  tube  E  until  it  had  just  passed  the  stopcock  F.  After  at- 
tachment had  been  made  to  the  pump  the  whole  was  evacuated  5 
hours  and  finally  at  a  pressure  of  1.2  mm.  the  capillary  at  II  was  sealed 
off  in  a  blowpipe  flame.  There  was  introduced  into  E  7.47  c.  c  X. 
T.  P.  of  oxygen  from  a  tank.  At  the  same  time  a  sample  of  the  same 
gas  was  taken  for  analysis,  and  found  to  contain  98.1  per  cent  of 
oxygen.  Upon  opening  the  cock  F  atmospheric  pressure  forced  the 
gas  over  into  the  tube  A.  The  mercury  behind  this  gas  wTas  allowed 
to  rise  until  it  had  entered  the  capillary  G.  As  close  to  this  mercury 
as  was  possible  G  was  then  fused  off  with  a  blowpipe.  There  was 
left  of  the  7.47  c.  c.  introduced  only  a  small  bubble  in  the  capillary, 
and  this  at  reduced  pressure.  After  6  days  at  25°  C.  the  tube  was 
opened  by  the  usual  method  and  the  gas  was  pumped  out  and  ana- 
lyzed, witli  the  following  result: 

Total  volume  of  gas  collected 11.  90 

Carbon  dioxid 10.  9G 

Oxygen 53 

Residue,  all  nitrogen 41 

From  the  percentage  composition  of  the  7.47  c.  c.  of  gas  added 
at  the  beginning  of  the  experiment  it  is  known  that  7.33  c.  c.  of 


20 


STUDY   OF    GASES   OF   EMMENTAL   CHEESE. 


oxygen  was  added.     At  the  end  of  the  experiment  there  remained 

only  0.53  c.  c.  of  oxygen.     There  must,  therefore,  have  been  6.80  c.  c. 

of  oxygen  absorbed,  or  0.239  c.  c.  per  gram  of  cheese. 

Such  an  active  absorption  of  oxygen  lends  itself  to  the  argument 

that  the  nitrogen  of  the  eyes  found  its  way  there  by  the  diffusion  in  of 
air.  But,  before  such  an  argument  can  be  con- 
sidered valid,  the  following  points  must  be  deter- 
mined :  First,  to  what  extent  is  cheese  permeable 
to  gases  in  general  and  nitrogen  in  particular  ? 
Second,  how  much  of  the  nitrogen  present  is  due 
to  nitrogen  dissolved  in  the  cheese  at  the  time  of 
manufacture?  Third,  what  evidences  are  there 
to  show  that  the  nitrogen  does  not  arise  in  situ 
from  bacterial  or  chemical  reactions  ? 


F 


THE  PERMEABILITY  OF  CHEESE  TO  GASES. 

After  various  unsuccessful  efforts  to  make  an 
impermeable  adhesive  that  would  stick  to 
cheese,  and  so  enable  a  slice  to  be  sealed  into 
a  diffusion  apparatus,  the  following  device  was 
made  (fig.  4)  : 

At  B  a  membrane  of  plaster  of  Paris  was 
formed  whose  strength  was  reenf orced  by  a  per- 
forated brass  plate  not  shown  in  the  diagram. 
This  membrane  was  desiccated  until  its  perme- 
ability was  high,  that  of  transfusion.10 

Most  of  the  air  was  forced  out  of  D  through 
the  membrane  and  E  by  raising  the  mercury. 
A  carefully  taken  disk  of  cheese  was  then 
placed  on  the  plaster  of  Paris  bed.  It  was 
gently  held  there  while  it  was  completely  cov- 
ered with  mercury.  Then,  by  lowering  F,  the 
space  in  D  was  left  under  greatly  reduced  pres- 
sure. This  caused  such  a  difference  in  pressure 
between  the  upper  and  lower  surfaces  of  the 
cheese  that  the  disk  was  held  firmly  against  the 
plaster  bed,  and  the  surrounding  mercury  was 
unable  to  float  it.  Preliminary  experiments 
showed  that  no  mercury  crept  between  the  disk  and  the  plaster, 
and  that  the  plaster  did  not  become  clogged  with  mercury  or  cheese. 
After  partial  vacuum  had  been  produced  in  D  a  few  moments  elapsed 
before  the  gas  retained  in  the  plaster  came  to  equilibrium.  When 
this  was  reached  the  mercury  was  carefully  withdrawn  from  the  top 
of  the  disk  of  cheese  until  the  surface  was  exposed.     The  mercury 


Fig.  4.— Device  for  ascertain- 
ing permeability  of  cheese  to 


1M.KM  I    Mill   IM      oltllll 


21 


left  at  the  ^i«  1«*  prevented  entrance  of  gas  there,    <>  thai   the  onlj 

|);l!  Il    he!  Wccll    t  lie  el  l;i  1 1 1 1  hts  A   ;ill«l    I  >   l>\    which  gttS  COtlld   «,!ili-r    I  >   WBi 

i  brough  t  he  cheese, 

The  disks  of  cheese  used  were  about  l  cm.  in  diameter  and  2  to 
2.5  mm.  thick.  They  were  taken  From  Bound  portions  of  freshly  cut 
cheese  by  means  of  a  cork  borer,  and  carefully  sectioned  with  a  sharp 
razor.  Every  precaution  was  used  in  cutting  and  handling  to  pre- 
vent distortion  and  breaking  of  the  texture.  In  one  case  the  <,v. p 
surface  w  as  t  liai  of  an  eve.  The  gas  \\  hose  diffusion  ii  was  desired  to 
study  was  flooded  into  the  chamber  A.  Willi  both  air  and  carbon 
dioxid  i  here  was  apparent  ly  no  diffusion  during  an  hour,  ei  en  I  bough 
the  pressure  in  l>  was  reduced  as  much  as  possible.  Longer  experi- 
ments were  not  practicable,  because  a  continuous  watch  bad  to  be 
kept  to  Bee  that  no  bubble4  of  air  entered  through  the  rubber  connecting 

tube  between  D  and  F  and  altered  the  volume  in  J).      With  a  trap  to 

prevent  such  a  source  of  error  the  same  impermeability  for  air  was 

observed  during  an  experiment  lasting  several  days. 

This  result  was  so  remarkable  that  it  was  tested  further  in  the 
following  manner:  Instead  of  the  parts  E,  D,  F  (fig.  4),  a  glass  tube 
led  from  B  to  a  mercury  pump.  With  the  cheese  slab  C  covered  with 
mercury  tin4  pump  was  operated  till  the  lowest  vacuum  which  could 
be  obtained  was  reached.  By  reason  of  the  gas  being  given  oil*  by  the 
cheese,  this  was  of  course  not  so  high  a  vacuum  as  the  pump  can 
produce.  When  the  vacuum  was  considered  sufficient  the  pump  was 
allowed  to  rest  in  order  to  discover  leakage,  and,  if  there  were  none, 
to  allow  the  residual  gas  to  distribute  itself  so  that  a  reliable  reading 
on  the  McLeod  gauge  could  be  made.  Then  the  mercury  was  care- 
fully withdrawn  from  the  top  of  the  cheese,  leaving  its  upper  surface 
exposed.  Entrance  of  gas  could  now  be  detected  by  the  McLeod 
gauge.     An  experiment  is  given  in  detail  below: 

[Disk  of  cheese  7  mm.  diameter,  2.5  mm.  thick,  taken  10.50  a.m.,  Dec.  19,1911, 15  mm. from  the  nearest  rind.] 


Time  of 

Pres- 

reading. 

sure. 

0.  TO. 

Mm. 

11.14 

0.075 

1 1 .  25 

.140 

11.30 

.150 

11.37 

.070 

11.40 

.070 

/>.  m. 

12.15 

.150 

1.15 

.270 

1.50 

.320 

2.15 

.350 

2.30 

.025 

3.10 

.060 

3.30 

.090 

3.35 

.025 

4.  in 

.050 

.065 

4.45 

.010 

o.  m.. 

9.30 

.430 

Apparatus  exhausted,  and,  with  cheese  covered  with  mercury,  pump  pressure  at 

Pump  res  ting 

Do 

Increase  in  pressure  assumed  to  be  due  to  gas  evolved  from  el  1 1 

After  7  minutes  pumping 

Cheese  exposed  to  air 

Do 

Do 

Do 

Do 

After  15  minutes  pumping 

Cheese  exposed  to  CO2 

Do 

After  5  minutes  pumping 

je  exposed  to  H2 

Do 

After  15  minutes  pumping 

Cheese  left  overnight  exposed  to  air 


22  STUDY   OF   GASES   OF   EMMENTAL   CHEESE. 

When  the  disk  of  cheese  and  the  mercury  were  removed  air  entered 
rapidly,  showing  that  the  plaster  had  not  become  plugged.  Further- 
more, there  was  no  evidence  of  mercury  having  crept  between  the 
cheese  and  the  plaster.  It  is  not  claimed  that  all  the  above  listed 
readings  on  the  McLeod  are  very  accurate,  since  the  readings  were 
sometimes  made  before  equilibrium  was  obtained.  All  that  was 
desired  was  the  order  of  magnitude.  Since  the  variation  in  tempera- 
ture during  the  experiment  was  only  between  the  extremes  17°  C. 
and  19°  C.  and  since  the  volume  of  the  pump,  gauge,  and  diffusion 
apparatus  was  found  to  be  159  c.  c,  we  may  calculate  from  pressures 
the  approximate  amount  of  gas  which  had  apparently  diffused  through 
the  cheese.  This  amounted  to  about  0.09  c.  c.  during  the  first  5  hours 
and  0.09  c.  c.  during  the  last  17  hours. 

Allowing  nothing  for  possible  small  leaks,  which  were  difficult  to 
avoid  in  the  delicate  manipulations  required,  the  observed  volume  of 
gas  indicates  a  very  remarkable  impermeability.  Practically  the 
same  result  was  obtained  with  a  disk  of  Cheddar  cheese  and  other 
samples  of  Swiss  cheese. 

The  question  at  once  arises,  How  to  explain  the  evolution  of  carbon 
dioxid  which  there  is  every  reason  to  suppose  does  diffuse  from  cheese  ? 
Van  Slyke  and  Hart 17  found  that  a  normal  Cheddar  cheese  evolved 
during  32  weeks  15.099  grams  of  carbon  dioxid.  Since  they  took 
care  to  exclude  surface  growths  of  molds,  it  seems  highly  improbable 
that  this  amount  of  carbon  dioxid  could  have  come  to  any  great 
extent  from  the  surface  layers  alone.  It  must  have  diffused  from 
the  interior  of  the  cheese  into  the  surrounding  bell  jar. 

The  following  explanation  will  doubtless  be  found  reasonable: 
Becquerel 4  found  that  when  the  tegument  of  peas  was  mounted  at 
the  end  of  a  barometer  tube,  and  a  partial  vacuum  of  5  to  10  mm. 
obtained  upon  the  one  side,  with  atmospheric  pressure  on  the  other, 
the  tegument  was  impermeable  to  gas  when  dry,  although  permeable 
when  moist.  In  so  far  as  the  tegument  of  peas  and  a  disk  of  cheese 
are  both  colloidal  they  may  be  compared.  In  the  present  experi- 
ments the  disks  of  cheese  dried  considerably  both  from  exposure  to 
gases  of  low  vapor  content  on  the  one  side  and  the  moisture  free 
vacuum  on  the  other.  By  analogy  with  Becquerel's  experiments 
one  would  expect  to  find  the  dry  cheese  more  or  less  impermeable. 
Reference  to  the  experiment  detailed  on  page  21  will  indeed  show  that 
the  permeability  decreased  as  the  time  of  the  experiment  increased, 
or,  in  other  terms,  as  the  cheese  became  drier.  Furthermore,  in 
an  experiment  in  which  the  exposed  surface  of  the  cheese  was  kept 
exposed  to  carbon  dioxid,  which  was  saturated  with  vapor,  1.04  c.  c. 
of  gas  was  found  to  have  passed  through  in  5  hours;  ten  times  as 
much  as  in  the  experiment  with  drying  cheese. 


I'l.KMl.  \l;l!   !  I  J      OF    (HUM.      N>   < 

It  therefore  seems  probable  thai  tin*  penneal>ilit\  i»f  rh«-«- »■  h. -.m  «-s 
is  due  to  the  diffusion  of  dissolved  ^iscs,  ami  that  .-is  the  free  solvent 
imea  more  and  more  attenuated  the  gai  is  more  and  more  unable 
to  find  its  way  through  the  geL 

Since  in  EmmentaJ  cheese  s  more  or  less  dry  rind  is  produced,  it 
seems  probable  thai  little  air  can  diffuse  into  the  cheese.  And  i 
the  fact  thai  in  the  manufacture  of  ( Iheddar  a  Less  dry  rind  as  well  as 
a  more  open  texture  is  produced,  ii  seems  probable  thai  escape  of 
carbon  dioxid  more  easily  occurs  in  this  type  than  in  the  Swiss  type 
of  cheese. 

It  must  be  remembered,  however,  thai  the  above  experiments  only 
cever  a  very  limited  time,  and  that,  even  were  the  permeabilit 
low  as  the  experiments  seem  to  show,  there  is  still  the  possibility  that 
nitrogen  may  make  its  way  slowly  through  the  gel  during  the  long 
period  of  ripening.  Possibly  more  extensive  investigation  would 
reveal  that  the  larger  percentages  of  nitrogen  found  in  the  eyes  of 
some  cheeses  are  proportional  to  the  age  of  the  cheeses.  Neverthe- 
less this  penetration  can  only  take  place  slowly. 

The  fact  that  penetration  of  air  is  so  slow,  together  with  the 
avidity  with  which  oxygen  is  absorbed,  only  tends  to  emphasize  the 
completeness  of  the  anaerohiosis  in  the  interior  of  the  cheese,  a  con- 
dition which  Troili-Peterson M  found  necessary  for  the  best  develop- 
ment of  the  propionic  bacteria. 

These  experiments  on  the  permeability  of  cheese  to  gases  make  it 
evident  that  in  pumping  the  gases  from  plugs  of  cheese  we  should  ex- 
pect the  gas  to  be  slowly  evolved.  Such  was,  indeed,  found  to  be  the 
case.  The  reason  for  this  was  not  fully  appreciated  at  the  time  the 
pumpings  were  made,  and  it  is  very  doubtful  if  all  the  occluded  gas 
completely  exhausted  even  after  20  hours  exposure  to  high 
vacuum.  Reference  to  the  experiment  with  plugs  of  cheese  kept  6 
days  in  vacuo  (p.  15)  reveals  the  interesting  fact  that  the  amount  of 
gas  evolved  per  gram  of  cheese  was  dependent  more  upon  the  state 
of  the  vacuum  than  upon  time.  This  is  illustrated  in  the  following 
statement,  in  which  the  figures  represent  cubic  centimeters  of  gas 
evolved  per  gram  of  cheese  per  hour: 


First  IS  hours 

Succeeding  6 
Last  18  hours 


.0015 
.0033 


0.0043 
.0014 

.0066 


During  the  middle  period,  of  course,  the  tubes  were  sealed,  and  the 
evolved  gas  increased  the  pressure.  Evidently,  then,  the  higher  the 
vacuum  to  which  the  sample  wras  subjected  the  more  rapidly  was  the 
gas  evolved,  indicating  that  a  considerable  proportion  of  the  gas  was 


24 


STUDY   OF    GASES   OF   EMMENTAL   CHEESE. 


M£/KMY 


A 


dissolved  or  occluded  gas  rather  than  that  formed  during  the  time 
of  the  experiment. 

It  may  also  be  true  that  there  is  loose  combination  of  carbon 
dioxid  with  inorganic  salts,  or  with  calcium  and  amino  bodies,  as  in 
the  carbo-amino  reaction,  and  that  the  stability  of  these  compounds 
is  a  function  of  the  imposed  pressure. 

NITROGEN  DISSOLVED  IN  CURD. 

Let  us  now  consider  how  much  of  the 
nitrogen  found  in  the  eyes  is  attributable 
to  nitrogen  occluded  in  the  original  curd. 
One  would  expect  the  curd  to  be  well 
aerated  by  the  vigorous  stirring  it  gets 
during  the  process  of  manufacture.  Mar- 
shall u  has  shown  that  aerated  milk  con- 
tains considerable  quantities  of  nitrogen, 
but,  unfortunately  for  the  purposes  de- 
sired, his  data  are  only  expressed  in  per- 
centage composition  and  not  very  defi- 
nitely in  cubic  centimeters  of  gas  per 
cubic  centimeter  of  milk. 

A  rough  approximation  of  the  amount 
of  nitrogen  occluded  in  the  curd  was 
obtained  in  the  following  way:  A  liter 
of  milk  was  treated  as  in  the  process  of 
making  Swiss  cheese.  When  the  curd 
had  reached  the  stage  when  it  was  suit- 
able to  hoop,  the  greater  part  of  the 
whey  was  decanted,  and  then  the  re- 
sidual whey  and  curd  were  poured  care- 
fully into  the  glass  cylinder  A,  figure  5 
(inverted).  As  the  curd  settled,  the 
overlying  whey  was  drawn  off  and  more 
of  the  mixture  poured  in.  This  was  re- 
peated until  the  tube  was  filled  with  curd 
grains  completely  surrounded  by  whey. 
The  rubber  stopper  was  then  forced  in. 
The  tube  was  next  inverted  to  the  posi- 
tion shown  in  the  figure,  and  the  mer- 
cury seal  stopcock  B  was  opened  to  re- 
lieve the  pressure.  The  rubber  stopper  was  then  forced  farther  in, 
and  the  whey  displaced  by  it  escaped  into  C.  By  covering  the 
stoppered  end  of  the  tube  with  rubber-rosin  cement  and  keeping 
it  under  mercury,  it  was  made  perfectly  gas  tight.  The  cock  B  was 
then  closed,  and,  after  the  surplus  whey  in  C  had  been  drained  out, 
the  apparatus  was  connected  to  the  vacuum  pump  in  the  usual  way. 


GLASS 

w 


Fig.  5.— Apparatus     for    determining 
amount  of  nitrogen  In  curd. 


NITR001  \    DISSOI  \  1  1>    IN    (l   i:D.  26 

When  the  pump  and  chamber  C  wm  completely  exhausted,  the 

cock  B  was  opened,  li  w;is  found  that  the  gas  expanding  in  A 
drove  the  whey  almost   completely  up  through  the  interstice!  ol  the 

curd  and  into  ('. 

An  interesting  point  was  observed.  Comparatively  little  of  the 
gas  came  from  the  whey,  while  the  major-  portion  came  From  the  curd 
particles.  Since  a  separation  oi'  whey  and  curd  was  accomplished, 
it  could  not  have  been  true  that  the  gas  evolved  from  the  curd  par- 
ticles originated  in  the  whey,  using  curd  particles  as  nuclei  for  the 
formation  of  bubbles.  Furthermore,  there  was  comparatively  Little 
frothing  oi'  the  whey  in  C,  most  of  the  gas  collected  having  bubbled 
through  C  from  A.  Examination  of  eurd  particles  will  show  why 
this  is  so;  for  they  have  adhering  to  them  minute  bubbles,  apparently 

froth  taken  up  during  the  stirring.  It  is  quite  evident  that  the  col- 
umn of  whey  in  C  through  which  the  gas  had  to  make  its  way  pre- 
vented a  very  complete  exhaustion.  Since  the  pumping  was  con- 
tinued several  hours  and  the  tube  then  allowed  to  stand  overnight 
before  the  final  pumping,  this  error  was  reduced  to  some  extent.  If 
occasion  arises  to  repeat  these  experiments  this  error  will  be  avoided. 

By  the  method  described,  1.35  c.  c.  of  gas  was  collected  in  one 
instance  and  0.86  c.  c.  in  another.  Of  this,  there  was  0.58  c.  c.  of 
nitrogen  in  one  case  and  0.39  c.  c.  in  the  other;  average,  0.5  c.  c. 
The  curd  was  roughly  estimated  to  represent  20  grams  of  cheese. 
Consequently  there  would  be  approximately  2.5  c.  c.  of  nitrogen 
per  100  grams  of  cheese.  How  this  nitrogen  would  partition  itself 
between  the  body  of  the  cheese  and  an  eye  is  a  question  wrhose  solu- 
tion would  be  mere  guesswork  without  further  data. 

While  the  2.5  c.  c.  per  100  grams  of  cheese  is  a  mere  approximation, 
and  a  figure  which  would  vary  not  only  wTith  the  extent  to  which  the 
curd  is  stirred,  but  also  with  the  form  of  the  curd  particles  and  their 
ability  to  absorb  foam,  nevertheless  it  is  sufficiently  accurate  to 
showr  that  a  large  part  of  the  free  nitrogen  found  in  cheese  comes  from 
occluded  air. 

DOES    NITROGEN    ORIGINATE    IN    SITU? 

The  question  of  whether  any  of  the  nitrogen  found  in  the  eyes  is 
set  free  in  situ  is  a  difficult  one  to  answer,  and  one  which  can  not  be 
definitely  answered  without  further  research.  From  the  following 
considerations,  however,  it  is  highly  probable  that  it  is  not  produced 
during  the  course  of  that  reaction  which  furnishes  the  gas  to  distend 
the  eyes.  In  those  experiments  in  which  samples  from  a  cheese  at 
the  period  of  its  maximum  eye  formation  were  held  in  vacuo,  the 
nitrogen  in  the  evolved  gas  steadily  and  rapidly  declined  in  per- 
centage, finally  reaching  almost  nothing.  This  indicates  that  the 
nitrogen  collected  was  simply  that  dissolved  in  the  cheese,  and  as 


26  STUDY  OF   GASES   OF   EMMENTAL   CHEESE. 

this  was  removed  there  was  no  evolution  of  free  nitrogen  to  take  its 
place,  such  as  occurred  in  the  case  of  the  carbon  dioxid. 

RELATION  BETWEEN  CARBON  DIOXID  AND  VOLATILE  ACIDS. 

The  results  of  the  whole  investigation  show  clearly  that  the  only 
gas  which  plays  an  important  role  in  the  formation  of  normal  eyes  is 
carbon  dioxid.  This  is  in  entire  harmony  with  the  assumption  which 
has  heretofore  been  accepted  as  a  fact  by  various  authors. 

It  remains  to  be  seen  whether  there  is  any  quantitative  relation 
between  the  amount  of  carbon  dioxid  evolved  and  that  called  for  by 
the  process  to  which  Von  Freudenreich  and  Jensen  ascribe  the  forma- 
tion of  eyes. 

A  study  of  the  volatile  fatty  acids  of  Emmental  cheese  by  Jensen  12 
disclosed  the  fact  that  they  are  chiefly  propionic  and  acetic,  and  that 
often  the  ratio  of  these  approximates  2:1. 

Fitz  7  had  previously  shown  that  certain  bacteria  are  capable  of 
producing  this  ratio  of  propionic  and  acetic  acids  from  lactic  acid, 
and  he  ascribed  to  their  action  the  equation : 

3C3H603=2C3H602+C2H402+C02+H20 
lactic  propionic        acetic 

Subsequently  Von  Freudenreich  and  Jensen8  isolated  from  Emmen- 
tal cheese  an  organism  which  did  ferment  lactates  according  to  the 
above  equation  of  Fitz,  and  whose  introduction  into  cheese  was  fol- 
lowed by  an  eye  formation  of  which  it  was  thought  to  be  the  cause. 

The  conclusion  seems  evident  that  here  is  an  organism  to  whose 
action  may  be  attributed  the  formation  of  normal  eyes. 

The  evidence  is  undoubtedly  the  clearest  that  has  yet  been  presented. 
There  are,  however,  one  or  two  points  which  will  bear  inspection 
before  the  theory  can  be  accepted  as  a  full  explanation. 

According  to  the  equation  of  Fitz  three  molecules  of  volatile  fatty 
acids  are  accompanied  by  the  liberation  of  one  molecule  of  carbon 
dioxid.  Consequently  it  can  be  shown  that  a  titer  of  1  c.  c.  of 
tenth-normal  alkali  for  these  volatile  fatty  acids  should  indicate  the 
liberation  of  0.74  c.  c.  of  carbon  dioxid  (N.  T.  P.).  If,  then,  it  is 
found  that  the  volatile  acids  from  100  grams  of  cheese  neutralize  100 
c.  c.  of  tenth-normal  alkali,  and  it  is  assumed  that  these  acids  are 
acetic  and  propionic  in  the  ratio  in  which  they  occur  in  Fitz's  equa- 
tion, we  would  have  liberated  74  c.  c.  of  carbon  dioxid  per  100  grams 
of  cheese. 

This  amount  of  gas  is  considerably  more  than  is  required  to  fill  the 
eyes,  but  the  question  remains  how  much  is  to  be  found  in  the  body 
of  the  cheese  itself. 

Reference  to  the  experiments  described  on  page  15  shows  that  at 
an  age  of  55  days  37.2  c.  c.  of  carbon  dioxid  per  100  grams  of  cheese 


i;i;i  \iio\    BBTW]  i  N    I   LBBOH    DIOXID   ani»   \<>i  mm  I   AOXM.      VJ  7 

were  collected  after  the  cheese  has  been  held  in  vacuo  one  week.  At 
the  time  of  the  experiment  it  was  thought  thai  tl  produced 

during  that  week.  After  the  study  which  shows  how  impermeable 
cheese  is,  this  view  had  to  be  modified,  for,  even  after  considerable 
pumping,  an  appreciable  quantity  of  gas  must  have  remained  and 
appeared  as  "'  evolved  "  gas  at  the  end  of  the  week.  En  order  to  make 
a  better  estimation  of  the  dissolved  gas,  plugs  of  this  Bame cheese  (at 
an  age  of  I  months)  were  sliced  into  thin  disks  to  facilitate  the  remoi  al 
of  dissolved  gas,  and  introduced  into  a  tube.  They  were  sealed  in 
with  the  usual  rubber  stopper  and  rubber-rosin  cement,  and  the  tube 
joined  to  the  mercury  pump.  After  evacuating  the  pump  the  con- 
necting cock  was  opened  and  the  disks  of  cheese  ei  acuated.  The  air 
Burrounding  them  in  the  tube  was  of  course  pumped  out  too.  The 
total  gas  thus  collected  after  ">  hours  continuous  pumping  contained 

17.i'.")   C.    C.    of   carbon  dioxid.      The  weight    of  cheese  was    1_>  grams. 

Hence,  there  were  collected  40.6  c.  c.  of  carbon  dioxid  per  LOO  grams  of 

cheese  (19  hours  later  0.9  C.  c.  of  carbon  dioxid  was  collected,  or  _M 
0,  c.  per  100  grams  of  cheese). 

A  duplicate  determination  gave  46.2  c.  c.  of  carbon  dioxid  per  100 
grams  of  cheese  (with  an  additional  1.03  c.  c.  per  100  grams  after  19 

horn's).  The  average  for  the  first  5  hours'  pumping  was  43.4  c.  c.  of 
carbon  dioxid  per  100  grams  of  cheese,  and  this  we  may  fairly  con- 
sider the  quantity  occluded  at  the  time  the  plugs  were  taken.  At 
the  same  age  (4  months)  the  volatile  fatty  acids  corresponded  to  40.9 
c.  c.  of  tenth-normal  alkali  per  100  grams  of  cheese. 

Similarly,  duplicate  determinations  of  dissolved  carbon  dioxid  and 
volatile  acids  in  an  excellent  imported  cheese  (No.  i)  gave  the  fol- 
lowing data:  Carbon  dioxid  per  100  grams,  67.8  c.  c.  and  54.8  c.  c, 
average  61.3  c.  c.  Total  volatile  fatty  acids  in  cubic  centimeters  of 
tenth-normal  alkali  per  100  grams  95.1  and  97.7,  average  90. 4. 

Assuming  that  all  the  volatile  fatty  acids  were  produced  in  strict 
accordance  with  the  equation  of  Fitz,  the  amount  of  these  acids  in 
the  first  cheese  indicates  that  there  had  been  liberated  30.6  c.  c.  of 
carbon  dioxid  against  43.4  c.  c.  found  occluded;  and  in  the  second 
cheese  the  liberation  of  71.3  c.  c.  of  carbon  dioxid  against  61.3  c.  c, 
found  occluded.  There  is  a  somewhat  striking  apparent  relation- 
ship in  this  data,  and  the  averages,  51.0  c.  c.  calculated,  against 
52.3  c.  c.  found,  are  in  such  close  agreement  that  they  are  tempting. 
A  little  consideration  will  show,  however,  that  this  agreement  may  be 
only  accidental.  At  the  time  these  analyses  were  made  each  of  the 
cheeses  had  probably  reached  a  state  of  little  activity.  The  volatile 
acids  represent  almost  entirety  the  total  amount  produced  in  the 
interior  from  which  the  samples  for  analyses  were  taken;  while,  if 
we  are  to  accept  the  results  on  Cheddar  cheese  by  Van  Slyke  and  Hart 
as  at  all  applicable  to  Eminent  al,  it  is  certain  that  a  considerable 


28  STUDY  OF   GASES   OF  EMMENTAL   CHEESE. 

quantity  of  carbon  dioxid  must  have  escaped  in  the  months  since 
manufacture.  Furthermore,  although  the  actual  volume  of  the  eyes 
represents  but  a  small  portion  of  the  gas  in  a  given  volume  of  cheese, 
the  normal  volume  of  this  gas  in  the  eyes  leaps  into  considerable 
significance  when  it  is  remembered  that  it  must  have  been  under 
considerable  pressure.  That  it  is  under  pressure  was  made  evident 
in  some  cases  by  its  vigorous  escape  when  using  the  puncturing 
apparatus  for  its  collection. 

Unfortunately  long  delay  in  obtaining  apparatus  suitable  for  a 
study  of  the  gas  escaping  from  cheese,  as  was  done  by  "Van  Slyke  and 
Hart  for  Cheddar,  have  made  it  impossible  to  present  any  data  on 
this  point.  As  before  mentioned,  the  data  on  carbon  dioxid  evolved 
from  plugs  of  cheeses  taken  at  the  height  of  their  gaseous  fermenta- 
tion and  kept  in  vacuo  a  week  is  complicated  by  the  fact  that  there 
was  probably  a  slow  jdelding  of  dissolved  gas  from  the  solid  plugs  as 
well  as  the  normal  production  of  gas.  Two  other  experiments,  how- 
ever, indicate  to  what  extent  carbon  dioxid  was  being  formed  during 
this  period  of  maximum  fermentation. 

Portions  of  cheese  W  2  from  regions  without  eyes  were  carefully 
selected  and  sealed  up  in  a  tube  as  described  on  page  14.  The  eye 
membranes  were  carefully  removed  from  a  large  number  of  eyes 
and  similarly  treated.  The  tubes  were  simultaneously  exhausted 
with  a  Bolt  wood  pump  for  several  hours.  Since  in  these  cases  the 
cheese  was  in  a  more  finely  divided  state,  it  is  reasonable  to  assume 
that  predissolved  gas  was  pretty  thoroughly  removed.  After  exhaus- 
tion, the  tubes  were  sealed  off  in  a  blowpipe  flame  and  held  at  25°  C. 
for  seven  days.     At  the  end  of  this  period  the  gas  was  collected: 

34  grams  eye  membranes  gave  14.95  c.  c.  of  gas,  99.3  per  cent  of  carbon  dioxid, 

or  44  c.  c.  per  100  grams. 
36  grams  from  regions  without  eyes  gave  10.06  c.  c.  of  gas,  98.2  per  cent  of  carbon 

dioxid,  or  28  c.  c.  per  100  grams. 

From  this  one  pair  of  experiments  it  is  not  advisable  to  claim  con- 
fidently that  the  eye  surfaces  always  produce  the  much  larger  quantity 
of  carbon  dioxid,  although  this  is  plainly  evident  in  the  above  case. 
The  significant  fact  is  that  such  a  large  quantity  was  produced  by 
each  region  in  the  period  of  only  one  week.  Of  course  it  may  be 
claimed  that  although  the  division  of  the  cheese  was  done  in  a  dust- 
free  room  and  with  sterile  instruments,  and  the  cheese  introduced 
into  sterile  tubes,  yet  the  long  manipulation  admitted  a  heavy 
reinoculation  by  bacteria,  and  that  these  produced  a  renewed  evolu- 
tion of  carbon  dioxid.  Such  an  argument  can  not  be  completely 
refuted,  but  the  probability  of  a  heavy  enough  infection  is  small. 
The  most  likely  source  of  carbon  dioxid  producing  infection  was  by 
molds,  but  these  could  not  have  grown  in  the  complete  anaerobic 
condition  in  which  the  cheese  quickly  found  itself. 


i;r.i  \  i  [OH    r.r.i  w  >  i  \    C  kSBOK    DIOXID  and  \«»i  \  i  n  I     \- 

The  following  experiment  serves  to  confirm  the  last.  Inf<>  i  iteri- 
lized  combustion  tube  were  quickly  slipped  plugs  of  chee 
with  sterile  instruments.  Each  end  of  the  tube  ^;is  guarded  with 
cotton  plugs.  Carbon  dioxid  free  air  was  then  passed  through,  and 
the  carbon  dioxid  evolved  from  the  cheese  absorbed  in  the  customary 
train  with  all  due  precautions  for  exact  estimation  of  carbon  dioxid. 
In  the  case  of  this  experiment  we  would  expect  ■  higher  amount  of 
carbon  dioxid,  since  there  would  be  collected  not  only  the  carbon 
dioxid  produced,  but  a  Large  portion  of  the  predissolved  carbon 
dioxid.    Such  was  found  to  be  the  cat 

One  hundred  and  five  grams  in  plugs  taken  from  cheese  W  l  when 
at  the  height  fermentation  gave: 

First  21  hours,  81.6  c.  c.  of  carbon  dioxid. 
Seo>ml  21  hours,  66.7  c.  c.  of  o&rbon  dioxid. 

Third  21  hours,  -1 1.5  c.  c.  of  carbon  dioxid. 
Fourth  21  hotu  C.  of  carbon  dioxid. 

The  increase  on  the  fourth  day  was  thought  to  be  duo  possibly  to 
growth  of  molds  with  which  Van  Siyke  and  Hart  found  difficulty  in 
their  work  on  Cheddar  cheese.  The  experiment  was  therefore  dis- 
continued, although  no  growth  was  visible. 

A  final  word  must  be  urged  against  the  too  liberal  use  of  the  equa- 
tion of  Fitz.  As  a  terse  representation  of  the  probable  relation  of 
the  end  products  the  equation  has  a  legitimate  use.  As  a  compre- 
hensive portraiture  it  is  colored  with  presumption.  The  literature  of 
fermentation  is  littered  with  equations,  two  or  three  members  of 
which  are  known  to  stand  in  certain  quantitative  relationships,  while 
the  other  members  are  given  values  which  fit.  This  stoichiometrical 
adjusting  is  particularly  true  of  the  gaseous  products.  One  has  only 
to  review  the  literature  on  the  gas  production  of  B.  coli  to  assure 
himself  of  the  fact. 

While  Von  Freudenreich  and  Jensen's  use  of  the  Fitz  equation  has 
been  interpreted  quite  rigidly  in  the  preceding  pages,  this  was  done 
simply  as  a  test.  From  this  basis  alone  one  can  not  reasonably  jump 
to  a  final  conclusion;  but  it  must  be  remembered  that  the  liberal  use 
made  of  the  Fitz  equation  was  generous  to  the  theory  of  Von  Freuden- 
reich and  Jensen  in  that  all  the  volatile  fatty  acids,  as  determined  by 
Jensen's  method,  were  assumed  to  have  been  produced  in  accordance 
with  this  equation. 

From  a  comprehensive  view  of  the  matter  it  appears  to  be  quite 
evident  that  the  theory  of  Von  Freudenreich  and  Jensen  is  not 
capable  of  accounting  for  all  the  carbon  dioxid  produced.  Indeed, 
it  is  not  necessary  nor  expected  that  it  should,  but  we  have  reached  a 
point  where  it  has  become  advisable  to  distinguish  between  a  primary 
and  a  secondary  cause  of  eye  formation,  and  to  at  least  define  clearly 
what  we  mean  when  we  attribute  to  any  organism  or  to  any  reaction 
the  function  of  forming  eyes. 


30  STUDY   OF    GASES   OF   EMMENTAL   CHEESE. 

Suppose  that  the  propionic  bacteria  are  active,  but  that  they  are 
never  sufficiently  localized  to  concentrate  carbon  dioxid  rapidly  enough 
at  one  point  to  produce  an  eye.  In  this  case  the  gas  would  be  more 
or  less  evenly  produced  throughout  the  body  of  the  cheese.  Now 
this  state  of  more  or  less  complete  saturation  of  the  body  with  car- 
bon dioxid  is  exactly  the  condition  necessary  for  the  most  advan- 
tageous eye  formation  by  any  other  reaction  which  may  follow,  else 
the  gas  evolved  at  a  point  would  be  largely  absorbed  and  its  inflating 
energy  dissipated.  Of  course  it  can  be  said  that  this  saturation 
proceeds  from  the  point  where  the  eye  is  formed,  and  that  the  delay 
observed  before  an  eye  commences  to  grow  represents  the  time 
necessary  to  effect  this  saturation. 

This,  however,  is  merely  presenting  the  other  horn  of  a  dilemma 
from  which  escape  is  possible  only  when  the  localization  of  the  propi- 
onic bacteria  is  conclusively  demonstrated.  Gorini 9  has  contended 
that  the  localization  of  colonies  may  often  be  of  as  great  importance 
as  their  isolation ;  and  it  is  interesting  to  note  that  he  found  no  cor- 
relation between  the  colonies  which  stained  on  his  sections  of  Grana 
cheese  and  the  gas  bubbles. 

If,  then,  we  distinguish  between  a  " saturating"  gas  production 
and  an  " inflating"  gas  production,  we  will  have  at  least  defined  a 
possibility  which  must  be  squarely  met,  and  a  Ivypothesis  which  may 
lead  to  a  differentiation  between  a  primary  and  a  secondary  cause  of 
eye  growth. 

The  favorable  results  obtained  with  cheese  inoculated  with  pro- 
pionic bacteria  indicate  that  they  may  play  an  important  role.  But 
is  this  role  primary  or  secondary  ?  Is  it  a  strictly  localized  action  or 
is  it  simply  the  provision  of  that  saturation  without  which  some 
primary  and  strictly  localized  reaction  would  be  without  avail  ?  The 
same  question  arises  in  the  case  of  the  glycerin-fermenting  bacteria 
to  which  Troili-Petersson  16  has  ascribed  an  important  role  in  the 
holing  of  Swedish  cheeses.  In  fact  experimental  cheesemaking  of  the 
past,  though  not  so  thoroughly  controlled  as  in  the  experiments  of 
Troili-Petersson  and  those  of  Yon  Freudenreich  and  Jensen,  bear 
evidence  that  any  one  of  a  number  of  gas-producing  bacteria  inay 
provide  the  saturation,  not  to  mention  those  reactions  which  Van 
Slyke  and  Hart 17  have  proposed  as  contributing  to  the  carbon  dioxid 
in  Cheddar  cheese.  On  the  other  hand,  any  one  of  these  may  be  the 
primary  "inflator"  and  the  other  the  secondary  "saturator." 

In  this  connection  it  may  be  of  interest  to  note  a  peculiar  phe- 
nomenon met  with  in  some  experimental  cheeses.  A  number  of 
these  made  with  "artificial"  rennet  by  Mr.  Doane  were  reported  in 
their  early  stages  to  have  begun  a  normal  eye  formation.  Seldom, 
however,  did  this  beginning  develop  into  a  normal  holing.  These 
cheeses  were  of  small  size,  and,  since  it  is  known  that  small-sized 
cheeses  for  some  reason  not  yet  clearly  defined  seldom  develop  large 


IfMABY.  1 

,  the  failure  in  these  c  general  principles  be  vaguely 

attributed  to  size.     However  thai  may  be,  it  was  found  upon  pump- 
ing out   tin*  dissolved  eras  that   tin-  amount    was  low.    The  three 


oheeses  examined  were  39   15,    ;'1   ii    I,  and   16   I    i.      See  Table  2, 

p.  ' 

It  is  well  known  from  the  work  of  Jensen  and  others  thai  the  bac- 
teria found  in  "natural"  rennet  are  of  ten  distinct  from  those  found 
in  *  'artificial"  rennet.  Since  the  cheeses  under  discussion  were  made 
with  the  latter,  is  it  not  possible  that  the  reaction  which  started  the 
eye  formation  was  rendered  inadequate  because  the  gas-producing 
propionic  bacteria,  which  might  have  saturated  the  cheese  with 
carbon  dioxid,  were  absent  I    That  the  observed  holes  were  truly 

the   beginnings   of   normal   eve-,    and    were    ool    B    pinhole    formation 

resulting  from  an  initial  gaseous  fermentation  of  sugar,  is  evinced 
by  the  fact  that  hydrogen  was  absent. 

Exhaustive  research  alone  can  unravel  this  tangle;  but  it  is  hoped 
that  the  present  investigation  has  provided  both  a  clearer  definition 
of  the  problem  and  a  sound  basis  of  fact. 

SUMMARY. 

1.  The  gases  of  normal  "eyes"  in  Emmcntal  cheese  are  exclu- 
sively carbon  dioxid  and  nitrogen,  and  of  these  only  the  carbon 
dioxid  is  of  significance. 

2.  The  nitrogen  accompanying  the  carbon  dioxid  in  normal  eyes  is 
that  of  air  originally  occluded  in  the  curd  at  the  time  of  manufacture. 

3.  There  sometimes  occurs  during  the  initial  fermentation  an  evo- 
lution of  gas  characterized  by  the  presence  of  hydrogen.  Tins  is 
believed  to  be  due  to  the  gaseous  fermentation  of  sugar. 

4.  The  hydrogen  from  such  an  initial  fermentation  may  sometimes 
linger  to  contaminate  the  gas  of  normal  eyes. 

5.  The  two  fermentations  are  distinct  and  are  characterized  by  their 
gaseous  products.  The  one  is  detrimental,  the  other  that  demanded 
of  a  good  Emmental  cheese. 

6.  High  oxygen-absorbing  power  combined  with  low  permeability 
of  the  cheese  to  air  render  the  interior  thoroughly  anaerobic,  and  conse- 
quently favorable  to  the  growth  of  anaerobic  bacteria. 

7.  A  comparison  between  the  amount  of  carbon  dioxid  evolved 
and  the  total  volatile  fatty  acids  shows  that  the  activity  of  the  pro- 
pionic bacteria  of  Von  Freudenreich  and  Jensen  is  not  sufficient  to 
account  for  all  the  carbon  dioxid  found. 

8.  It  was  found  that  cheese  is  capable  of  retaining  a  very  large 
amount  of  carbon  dioxid. 

9.  The  possibility  is  suggested  that  there  are  two  phases  in  the  for- 
mation of  normal  eyes,  a  saturation  of  the  body  with  carbon  dioxid, 
and  an  inflation  of  eyes;  and  the  bearing  of  this  hypothesis  on  the 
production  of  gas  by  a  specific  cause  is  discussed. 


32  STUDY  OF   GASES   OF  EMMENTAL   CHEESE. 

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